Dataset Information

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.

ABSTRACT: Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals.

SUBMITTER: Brown KJ

PROVIDER: S-EPMC3642779 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.

Brown Kristy J KJ Marathi Ramya R Fiorillo Alyson A AA Ciccimaro Eugene F EF Sharma Seema S Rowlands David S DS Rayavarapu Sree S Nagaraju Kanneboyina K Hoffman Eric P EP Hathout Yetrib Y

Journal of bioanalysis & biomedicine 20121201

Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based app ...[more]

PMID: 23646235

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Project description:BackgroundWe are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells.AimsOur aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells.MethodsSkeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression.ResultsCell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs.ConclusionsEctopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.

Dataset Information

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.

Publications

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry.

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